(1) Field of the Invention
The present invention relates to a method of obtaining a fluorescent image, and an apparatus for carrying out the method; and a method of detecting a fluorescence-labeled DNA so as to inspect the DNA, and an apparatus for carrying out the method.
Furthermore, the present invention relates to a method of detecting or inspecting a matter which emits fluoresce by itself, or a fluorescence-labeled matter, in particular, a fluorescence-labeled DNA, and an apparatus for carrying out the method. Particularly, the present invention relates to a high-speed method of detecting or inspecting a fluorescent material as arranged in the form of a bead-array or a dot-array with high sensitivity and in a wide dynamic range, and an apparatus for carrying out the method.
(2) Description of the Related Art
An apparatus for shifting a position to be dotted on a glass substrate by means of a spotter according to the type of a probe DNA; and/or an apparatus for reading the intensity of fluorescence of samples which are provided by hybridizing a fluorescence-labeled probe DNA with what is called a DNA micro-array as obtained according to a photolithography technology have been employed in this field. With such an apparatus, a procedure comprising the steps of: irradiating a fluorescence-labeled probe DNA with an exciting laser spot beam having a certain fixed intensity, detecting a fluorescent light as generated from the probe DNA by means of a photo-multiplier, and determining the intensity of fluorescence from the detected signal is sequentially carried out for all the samples.
When such an apparatus is used for the expression and/or analysis of a DNA in tissue, whose object is to inspect the DNA, the ratio of concentration between target mRNAs or between target cDNAs which are the copies of the mRNAs may be larger than 1:10,000. Even in such a case, in order to precisely detect the targets, it is very important to detect the same in a wide dynamic range.
However, the prior method of detection mentioned above has, for example, the problem that it is not easy to detect the same in a wide dynamic range, and furthermore remarkably much time is required.
In addition, conventionally, as a method of detecting samples wherein fluorescent materials or fluorescence-labeled DNAs are arranged in the form of a bead array or a dot array, a method comprising the steps of: forming an exciting laser light into one spot, relatively scanning samples with the exciting laser spot light, and detecting the resultant fluorescent lights has been used. Besides, a method comprising the steps of: area-irradiating the wide area of a sample with an exciting light, and detecting the resultant fluorescent lights by means of a two-dimensional CCD or the like.
When the one spot light mentioned above is irradiated so as to relatively scan the samples with the exciting laser spot light for detection, since samples in the form of bead or dot array are scanned all over the surface thereof, the rate of time as substantially and effectively used for detecting fluorescent lights is very small as explained below. That is to say, when the diameter of a bead or dot is represented by “D”, and the pitch between the beads or dots is represented by “P”, if the array is in the form of an in-line arrangement, the rate of time as effectively used for detecting fluorescent lights during the scanning is πD2/4P2. Then, for example, if the ratio of D to P is 1:2, the rate of time is 19.6%, and even if the ratio is 1:1.5, the rate of time is 34.9%, which means that more than a half of the time is not effectively used for detection.
Furthermore, since one spot light is irradiated for detection, when samples consist of a lot of beads or dots, much time is required for detecting fluorescent lights. Therefore it is difficult to detect fluorescent lights at a high speed. In addition, when it is intended to detect fluorescent lights with high sensitivity and in a wide dynamic range, since time for exciting one bead or dot is to some extent required, the sensitivity and the dynamic range have to be sacrificed for achieving high-speed detection.
Similarly, when the area-irradiation and the two-dimensional detection are employed as mentioned above, the above-mentioned useless time is required, and thus the dynamic range have to be sacrificed for achieving high-speed detection.